(1) State of The Invention
This invention relates to a method of generating light chemically (chemiluminescence) in an assay on a surface such as a membrane by the action of a peroxidase enzyme with an oxidant such as hydrogen peroxide on a group of hydroxyaryl cyclic diacylhydrazides. The invention also relates to a method of greatly increasing the amount of chemiluminescence produced from this process by the use of specific enhancers and by conducting the reaction on a membrane. The invention also relates to the use of this method to detect the peroxidase enzyme. Further, the invention relates to the use of the method to detect and quantitate various biological molecules. For example, the method may be used to detect haptens, antigens and antibodies by the technique of immunoassay, proteins by Western blotting, DNA and RNA by Southern and Northern blotting, respectively. The method may also be used to detect RNA and DNA in dot blots, DNA in sequencing applications, and DNA in DNA probe assays.
(2) Prior Art
a. Chemiluminescent Oxidation of Hydroxyaryl Cyclic Diacylhydrazides.
A series of reports has appeared concerning the chemiluminescent reactions of 3- or 4-hydroxyphthalhydrazide with different chemical oxidizing agents (I. E. Kalinichenko, A. T. Pilipenko, V. A. Barovskii, Ukr. Khim. Zh. (Russ. Ed.), 43(10), 1102-6 (1977); Kalinichenko, I. E., Barovskii, V. A., Pilipenko, A. T., Ukr. Khim. Zh. (Russ. Ed.), 44(7), 748-52 (1978); A. T. Pilipenko, V. A. Barovskii, I.E. Kalinichenko, Zh. Anal. Khim., 33(10), 1880-4 (1978); I. E. Kalinichenko, V. A. Barovskii, Ukr. Khim. Zh. (Russ. Ed.), 45(1), 58-62 (1979); I. E. Kalinichenko, T. M. Tkachuk, A. T. Pilipenko, Zh. Anal. Khim., 39(7), 1281-4 (1984)). Gundermann reports the chemiluminescent oxidation of 7-hydroxynaphthalene-l,2-dicarboxylic hydrazide in aqueous solution with hydrogen peroxide and a metal catalyst (K.-D. Gundermann, W. Horstmann, G. Bergman, Lieb. Ann. der Chem, 684, 127-141 (1965)). No reports are known concerning the use of hydroxyaryl cyclic diacylhydrazides with a peroxidase enzyme or any enzyme to generate chemiluminescence. Further, there are no known reports of the use of hydroxyaryl cyclic diacylhydrazides to generate chemiluminescence for the detection of biological compounds.
b. Chemiluminescent Oxidation of Luminol and Related Compounds.
Aminoaryl cyclic diacylhydrazides such as luminol and isoluminol react with H.sub.2 O.sub.2 and a peroxidase enzyme catalyst (such as horseradish peroxidase, HRP) under basic conditions with emission of light. The reaction is also catalyzed by small amounts of several metal ions including Fe(III), Cu(II) and Cr(III) or iron-containing organic compounds (e.g. R. B. Brundett, D. F. Roswell, E. H. White, J. Am. Chem. Soc., 94, 7536 (1972)). This reaction has been used as the basis for analytical methods for the detection of H.sub.2 O.sub.2 and for metal ions. Luminol and isoluminol may be directly conjugated to a species to be detected. The first chemiluminescent immunoassay using luminol as a label was reported by Schroeder for an assay of biotin. (H. R. Schroeder, P. 0. Vogelhut, R. J. Carrico, R. C. Boguslaski, R. T. Buckler, Anal. Chem. 48, 1933 (1976)). Several applications of the use of luminol derivatives as labels have been reported since then (H. R. Schroeder in Luminescent Immunoassays: Perspectives in Endocrinology and Clinical Chemistry, M. Serio and M. Pazzagli, Eds., Raven Press, New York, pp 129-146 (1982); M. Pazzagli, G. Messeri, A. L. Caldini, G. Monetti, G. Martinazzo, M. Serio, J. Steroid Biochem., 19, 407 (1983); J. DeBoever, F. Kohen and D. Vandekerckhove in Bioluminescence and Chemiluminescence New Perspectives, J. Scholmerich, et al., Eds., J. Wiley & Sons, Chichester , pp 257-260 (1987)). Various enhancers have also been employed in conjunction with horseradish peroxidase in the oxidation of luminol to increase the intensity of light emitted. These include, for example, D-luciferin (T. P. Whitehead, G. H. Thorpe, T. J. Carter, C. Groucutt, L. J. Kricka, Nature, 305, 158 (1983)) and p-iodophenol and p-phenylphenol (G. H. Thorpe, L. J. Kricka, S. B. Mosely, T. P. Whitehead, Clin. Chem., 31, 1335 (1985)).
c. Enzyme-Catalyzed Chemiluminescent Reactions
(1) Enzymatic Generation of Hydrogen Peroxide. Various enzymatic reaction schemes are known which produce hydrogen peroxide. The generated hydrogen peroxide can, in turn, be used to oxidize a compound which emits light. For example, glucose oxidase reacts with O.sub.2 and sucrose to produce H.sub.2 O.sub.2. Similarly, amino acid oxidase and cholesterol oxidase react with amino acids or cholesterol, respectively, and O.sub.2 to produce H.sub.2 O.sub.2. Examples of compounds which are oxidized by hydrogen peroxide to produce light are luminol and isoluminol, lucigenin, esters of N-methylacridine and esters or amides of oxalic acid. Glucose-6-phosphate dehydrogenase and galactose-6-phosphate dehydrogenase have been used to produce H.sub.2 O.sub.2 indirectly by reduction of oxygen through an electron-relay system (K. Tanabe, T. Kawasaki, M. Maeda, A. Tsuji, M. Yabuuchi, Bunseki Kagaku, 36, 82 (1987); and A. Tsuji, M. Maeda, H. Arakawa, Anal. Sci., 5 497 (1989)).
(2) Enzymatic Deprotection of a Luminol Derivative. The compound o-aminophthalhydrazide-N- acetyl-.beta.-D-glucosaminide (luminol-NAG) and 4'-(6'-diethylaminobenzofuranyl)phthalhydrazide-N-acetyl-.beta.-D-glucosam inide are substrates for the enzyme N-acetyl-.beta.-D-glucosaminidase which serve as a masked form of luminol. Upon action of the enzyme on these substrates, luminol or a luminol derivative are liberated which may be detected as described above (K. Sasamoto, Y. Ohkura, Chem. Pharm. Bull. 38(5), 1323 (1990); K. Sasamoto, Y. Ohkura, Chem. Pharm. Bull. 39(2), 411 (1991)).
d. Use of Chemiluminescence in DNA Hybridization assays.
Biological assays such as enzyme immunoassays and DNA probe assays involving enzymes utilize a wide variety of substrates which either form a color (chromogenic), become fluorescent (fluorogenic) or emit light (chemiluminogenic) upon reaction with the enzyme. Of these three choices, chemiluminescence offers the greatest sensitivity. In an assay, the enzyme (reporter enzyme) is conjugated or bound to the molecule to be detected or to some other substance capable of selectively binding or associating with the molecule to be detected. Once the bound reporter enzyme is separated from unbound enzyme, a substrate is provided with which the reporter enzyme generates a signal. The enzyme horseradish peroxidase has been widely used in enzyme immunoassays and DNA hybridization assays with chemiluminescent detection using luminol or isoluminol as substrate (T. P. Whitehead, G. H. Thorpe, T. J. Carter, C. Groucutt, L. J. Kricka, Nature, 305, 158 (1983); G. H. Thorpe, L. J. Kricka, S. B. Mosely, T. P. Whitehead, Clin. Chem., 31, 1335 (1985); G. H. Thorpe, S. B. Mosely, L. J. Kricka, R. A. Stott, T. P. Whitehead, Anal. Chim. Acta, 170, 107 (1985); J. A. Matthews, A. Batki, C Hynds, L. J. Kricka, Anal. Biochem., 151, 205, (1985)). Commercially available kits for conjugation of HRP with enhanced luminol chemiluminescent detection are sold under the tradename "AMERLITE.TM." (Amersham International, PLC., Amersham, England).
OBJECTS
It is therefore an object of the present invention to provide a method and hydroxyaryl cyclic diacylhydrazides for use in generating chemiluminescence by the action of a peroxidase enzyme and a phenol enhancer for the detection of biological materials and compounds. It is also an object of the present invention to provide a method and kit using hydroxyaryl cyclic diacylhydrazides on membranes for generating chemiluminescence by the action of a peroxidase enzyme and a phenol enhancer for the detection of peroxidase enzymes and enzyme-conjugates. Additionally, it is an object of the present invention to provide a method and kit using hydroxyaryl cyclic diacylhydrazides on surfaces such as membranes for generating chemiluminescence by the action of a peroxidase enzyme and a phenol enhancer for detection of nucleic acid assays in solution and on surfaces. Further, it is an object of the present invention to provide a method and kit using hydroxyaryl cyclic diacylhydrazides for generating chemiluminescence by the action of a peroxidase enzyme and a phenol enhancer, for detection of proteins in Western blots and nucleic acids in Northern blots, Southern blots and other DNA hybridization assays.